This application is designed to facilitate the training and career development of the applicant, whose goal is to be an independent investigator. The proposed studies provide the applicant with the opportunity to build on existing knowledge and learn a wide range of cell biology, biochemistry, and animal techniques that will allow her to develop expertise in metabolism and obesity research. The research plan includes a novel and significant project proposal that provides opportunities to acquire new laboratory techniques. The training plan also provides opportunities to further hone scientific writing and presentation skills and increase scientific independence. Type 2 diabetes is characterized by impaired insulin action in liver, muscle, and adipose tissue. Obesity, insulin resistance, and their related disorders are a growing health concern reaching epidemic proportions, making the need to develop effective treatments highly important. The long term objective of the proposal is to determine the mechanisms involved in the pathogenesis of insulin resistance and diabetes. Retinol binding protein 4 (RBP4) is elevated in rodents and humans with obesity and insulin resistance. Furthermore, elevating RBP4, either genetically or by injections, causes insulin resistance, glucose intolerance, and impaired insulin signaling in skeletal muscle of rodents. Obesity and insulin resistance are associated with inflammation. Preliminary data show that RBP4 stimulation of macrophages causes cytokine release and insulin resistance in co-cultured adipocytes. This grant will test the hypothesis that RBP4 causes insulin resistance in part by stimulating a macrophage-mediated inflammatory response in adipose tissue. Cell culture assays, a high-throughput cell-based RNAi screen, and animal models will be used. Specific Aim #1 is to investigate the cellular mechanisms involved in RBP4-dependent activation of the inflammatory response. It will be determined if RBP4-stimulation of cytokine release from macrophages and the resulting insulin resistance in adipocytes are dependent on the RBP4 receptor, STRA6 (Stimulated by Retinoic Acid 6) by depletion of STRA6 in macrophages. The role of Toll-like Receptor 4 (TLR4) in RBP4-induced cytokine release from macrophages and the resulting insulin resistance in adipocytes will also be determined. Cellular pathways involved in the RBP4-induced inflammatory response in macrophages will be identified using a high-throughput RNAi screen in collaboration with the Broad Institute. Specific Aim #2 is to determine if RBP4 causes insulin resistance by increasing adipose tissue macrophage infiltration and cytokine release in vivo. Normal mice will be injected with RBP4 to determine if elevated levels are sufficient to initiate adipose tissue inflammation. Serum RBP4 levels will be lowered in obese mice to determine if elevated serum RBP4 levels are necessary for obesity-induced adipose tissue inflammation. These studies will elucidate mechanisms for RBP4 induced insulin resistance and may identify novel therapeutic targets to prevent or treat type 2 diabetes. PUBLIC HEALTH RELEVANCE: The proposed experiments will expand our understanding of mechanisms that contribute to causing obesity, insulin resistance, and type 2 diabetes. Understanding the mechanisms by which retinol binding protein 4 contributes to the development of insulin resistance and adipose tissue inflammation will guide the design of new therapeutic targets to prevent insulin resistance and treat type 2 diabetes.